Inhibited expression of pd-l1 and enhanced expression of pd-1

ABSTRACT

Processes for treating an autoimmune disease or condition by decreasing expression of a CD274 gene and/or a PD-L1 protein and increasing expression of a PDCD1 gene and/or a PD-1 protein in a subject in need of such expression changes are provided. The processes include administering to the subject a composition including at least 0.5% of cinnamtannin D-1 and/or cinnamtannin B-1. The processes further include decreasing expression of a CD274 gene and/or a PD-L1 protein and increasing expression of a PDCD1 gene and/or a PD-1 protein in the subject by the step of administering at the desired concentration or for the desired time wherein the composition may increase or decrease the relative expression levels of the genes or proteins while simultaneously ameliorating, preventing, or modulating a cardiac event in the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/128,786 filed Sep. 12, 2018, and depends from and claims priority toU.S. Provisional Application No. 62/557,933 filed Sep. 13, 2017, theentire contents of each of which are incorporated herein by reference.

FIELD

The present disclosure relates to the use of Type A polymers, includingA-Type doubly linked procyanidin oligomers of the catechins and/orepicatechins, and methods for promoting decreased expression of a CD274gene or a PD-L1 protein and enhanced expression of a PDCD1 gene or aPD-1 protein in a subject.

BACKGROUND

Dysregulated cellular function underlies many pathological conditions.Identification of molecular effectors of proper cell function along withmethods of preventing or reversing cell dysfunction are required forpromoting or maintaining proper or enhanced cellular function.Dysregulated gene expression is implicated in a variety of diseases suchas cancer, diabetes, obesity, cardiovascular diseases andneurodegeneration. Discerning which molecular targets are most directlyassociated with a disease or condition is paramount to treating orpreventing the disease and condition.

The programmed death 1 (PD-1) receptor and PD-1 ligands 1 and 2 (PD-L1,PD-L2) play integral roles in immune regulation. Expressed on activatedT cells, PD-1 is activated by PD-L1 and PD-L2 expressed by stromalcells, tumor cells, or both, initiating T-cell death and localizedimmune suppression, potentially providing an immune-tolerant environmentfor tumor development and growth. Conversely, inhibition of thisinteraction can enhance local T-cell responses and mediate antitumoractivity in nonclinical animal models. In the clinical setting,treatment with antibodies that block the PD-1-PD-L1 interaction havebeen reported to produce objective response rates of 7% to 38% inpatients with advanced or metastatic solid tumors, with tolerable safetyprofiles.

The programmed death 1 (PD-1) receptor and programmed death-ligand 1(PD-L1) play integral roles in immune regulation. PD-L1 (also calledB7-H1 or CD274) is a 290 amino acid protein receptor ligand encoded bythe CD274 gene and is expressed widely on both lymphoid and non-lymphoidtissues such as CD4 and CD8 T-cells, macrophage lineage cells,peripheral tissues as well as on tumor cells, and virally-infected cells(Dong et al. 1999, Nature Med.). PD-L1 binds to receptors PD-1 and B7-1which belong to the CD28/CTLA-4 (cytotoxic T lymphocyte antigen)/ICOS(inducible co-stimulator) family of T-cell co-inhibitory receptors andattenuates the immune response by inhibiting T-cell activation. PD-L1binding to PD-1 or B7-1 results in decreased T-cell proliferation andcytokine secretion, compromising humoral and cellular immune responsesin diseases such as cancer, and viral infection.

The expression of PD-L1 on tumor cells and virally-infected cells isexploited by tumors and chronic viral infections to evade immuneresponse. PD-L1 is expressed on a wide variety of tumors and studies onanimal models have shown that PD-L1 on tumors inhibits T-cell activationand lysis of tumor cells and may lead to increased death oftumor-specific T-cells. In chronic viral infections, PD-L1 expressed onvirally-infected cells binds to PD-1 on virus-specific T-cells and theseT-cells become “exhausted” with loss of effector functions andproliferative capacity. The PD-1-PD-L1 system also plays an importantrole in induced T-regulatory cell development and in sustaining Tregulatory cell function. Accordingly, as PD-L1 plays an important rolein tumor immunity and infectious immunity, a composition that decreasesexpression of a CD274 gene or a PD-L1 protein is an ideal target forimmunotherapy.

However, cardiac side effects have been observed in treatments thatresult in the decrease of PD-L1 expression. For example, in one study,targeted inhibition of PD-L1 with pembrolizumab was associated withacute heart failure due to autoimmune myocarditis (Laubli et al., Acuteheart failure due to autoimmune myocarditis under pembrolizumabtreatment for metastatic melanoma, Journal for ImmunoTherapy of Cancer(2015) 3:11).

PD-1 is an immunosuppressive receptor belonging to an immunoglobulinfamily and is encoded by the PDCD1 gene. PD-1 is a molecule having afunction of suppressing the immune activation signals of T-cellsactivated by stimulation through an antigen receptor. For example,analysis of PD-1 knock-out mice demonstrates that PD-1 signals playimportant roles in suppression of autoimmune diseases such as autoimmunedilated cardiomyopathy, lupus-like syndrome, autoimmuneencephalomyelitis, systemic lupus erythematosus, graft-versus-hostdisease, type I diabetes mellitus, and rheumatoid arthritis.Accordingly, as PD-1 plays an important role in autoimmune diseases, acomposition that enhances expression of a PDCD1 gene or a PD-1 proteinis an ideal target for immunotherapy.

As such, there exists a need for compositions and methods of decreasingexpression of a CD274 gene or a PD-L1 protein while increasingexpression of a PDCD1 gene or a PD-1 protein in a subject.

SUMMARY

It is understood that both the following summary and the detaileddescription are exemplary and explanatory and are intended to providefurther explanation of the disclosure as claimed. Neither the summarynor the description that follows is intended to define or limit thescope of the disclosure to the particular features mentioned in thesummary or description.

One object is to provide a method for altering levels of a CD274 geneproduct(s) or a PD-L1 protein and a PDCD1 gene expression product(s) ora PD-1 protein. Such expression can be used restore abnormal expressionof these expression products in a subject.

This object is achieved in the present disclosure that providesprocesses for decreasing expression of a CD274 gene or a PD-L1 proteinwhile increasing expression of a PDCD1 gene or a PD-1 protein in asubject. The processes include administering to the subject acomposition comprising at least 0.5% Type-A polymers by weight. TheType-A polymers include A-Type doubly linked procyanidin oligomers ofthe catechins and/or epicatechins. In certain aspects, the A-Type doublylinked procyanidin oligomers of the catechins and/or epicatechinsinclude cinnamtannin D-1 and/or cinnamtannin B-1. Further processesinclude decreasing expression of a CD274 gene or a PD-L1 protein whileincreasing expression of a PDCD1 gene or a PD-1 protein in said subjectby the step of administering.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the illustrative examples in the drawings:

FIG. 1 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with isolated doubly linkedtype-A polymers from an extract of cinnamon at a concentration of at 0μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, or 50 μg/ml for 24 hours at 37° C.where results are expressed as the mean±SD of five to ten random fieldswith approximately similar density of cells in each field in each plate;

FIG. 2 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with cinnamtannin B-1 at aconcentration of at 0 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, or 20 μg/mlfor 24 hours at 37° C. where results are expressed as the mean±SD offive to ten random fields with approximately similar density of cells ineach field in each plate;

FIG. 3 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with cinnamtannin D-1 at aconcentration of at 0 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, or 20 μg/mlfor 24 hours at 37° C. where results are expressed as the mean±SD offive to ten random fields with approximately similar density of cells ineach field in each plate;

FIG. 4 graphically depicts the effects on LPS-induced overexpression ofPDCD-1 as quantitative densitometry results of immunofluorescencestudies for human macrophage-like THP-1 cells treated with LPS at 10μg/ml and isolated doubly linked type-A polymers from an extract ofcinnamon at a concentration of at 0 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml,or 50 μg/ml for 24 hours at 37° C. where results are expressed as themean±SD of five to ten random fields with approximately similar densityof cells in each field in each plate;

FIG. 5 graphically depicts the effects on LPS-induced overexpression ofPDCD-1 as quantitative densitometry results of immunofluorescencestudies for human macrophage-like THP-1 cells treated with LPS at 10μg/ml and cinnamtannin D-1 at a concentration of at 0 μg/ml, 2.5 μg/ml,5 μg/ml, 10 μg/ml, or 25 μg/ml for 24 hours at 37° C. where results areexpressed as the mean±SD of five to ten random fields with approximatelysimilar density of cells in each field in each plate;

FIG. 6 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with garlic acid at aconcentration of at 0 μg/ml, 10 μg/ml, 20 μg/ml, or 50 μg/ml for 24hours at 37° C. where results are expressed as the mean±SD of five toten random fields with approximately similar density of cells in eachfield in each plate;

FIG. 7 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with rutin (the glycosidecombining the flavonol quercetin and the disaccharide rutinose) at aconcentration of at 0 μg/ml, 10 μg/ml, 20 μg/ml, or 50 μg/ml for 24hours at 37° C. where results are expressed as the mean±SD of five toten random fields with approximately similar density of cells in eachfield in each plate;

FIG. 8 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with quercetin at aconcentration of at 0 μg/ml, 10 μg/ml, 20 μg/ml, or 50 μg/ml for 24hours at 37° C. where results are expressed as the mean±SD of five toten random fields with approximately similar density of cells in eachfield in each plate;

FIG. 9 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with olive leaf extract at aconcentration of at 0 μg/ml, 10 μg/ml, 50 μg/ml, or 100 μg/ml for 24hours at 37° C. where results are expressed as the mean±SD of five toten random fields with approximately similar density of cells in eachfield in each plate;

FIG. 10 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with rosemary extract at aconcentration of at 0 μg/ml, 10 μg/ml, 50 μg/ml, or 100 μg/ml for 24hours at 37° C. where results are expressed as the mean±SD of five toten random fields with approximately similar density of cells in eachfield in each plate;

FIG. 11 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with a black chokeberryextract from the Aronia (CellBerry®) at a concentration of at 0 μg/ml,10 μg/ml, 20 μg/ml, or 100 μg/ml for 24 hours at 37° C. where resultsare expressed as the mean±SD of five to ten random fields withapproximately similar density of cells in each field in each plate;

FIG. 12 graphically depicts the effects on PDCD-1 expression asquantitative densitometry results of immunofluorescence studies forhuman macrophage-like THP-1 cells treated with curcumin extract at aconcentration of at 0 μg/ml, 5 μg/ml, 10 μg/ml, or 50 μg/ml for 24 hoursat 37° C. where results are expressed as the mean±SD of five to tenrandom fields with approximately similar density of cells in each fieldin each plate;

FIG. 13 graphically depicts the effects on PD-L1 expression asquantitative densitometry results of immunofluorescence studies for ratC6 glioma cells treated with cinnamtannin D1 at a concentration of at 0μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, or 50 μg/ml for 24 hours at 37° C.where results are expressed as the mean±SD of five to ten random fieldswith approximately similar density of cells in each field in each plate;

FIG. 14 graphically depicts the effects on PD-L1 expression asquantitative densitometry results of immunofluorescence studies for ratC6 glioma cells treated with cinnamtannin B1 at a concentration of at 0μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, or 50 μg/ml for 24 hours at 37° C.where results are expressed as the mean±SD of five to ten random fieldswith approximately similar density of cells in each field in each plate;

FIG. 15 graphically depicts the effects on TNF-α-induced overexpressionof PD-L1 as quantitative densitometry results of immunofluorescencestudies for rat C6 glioma cells treated with TNF-α at 10 ng/ml andcinnamtannin D1 at a concentration of at 0 μg/ml, 10 μg/ml, 20 μg/ml, or50 μg/ml for 24 hours at 37° C. where results are expressed as themean±SD of five to ten random fields with approximately similar densityof cells in each field in each plate;

FIG. 16 graphically depicts the effects on LPS-induced overexpression ofPD-L1 as quantitative densitometry results of immunofluorescence studiesfor rat C6 glioma cells treated with LPS at 10 μg/ml and cinnamtannin D1at a concentration of at 0 μg/ml, 10 μg/ml, or 50 μg/ml for 24 hours at37° C. where results are expressed as the mean±SD of five to ten randomfields with approximately similar density of cells in each field in eachplate; and

FIG. 17 graphically depicts the effects on PD-L1 expression asquantitative densitometry results of immunofluorescence studies for ratC6 glioma cells treated with a purified type-A polymer material with atleast 3% by weight type-A polymers tested at a concentration of at 0μg/ml, 5 μg/ml, 10 μg/ml, 50 μg/ml, or 100 μg/ml for 24 hours at 37° C.where results are expressed as the mean±SD of five to ten random fieldswith approximately similar density of cells in each field in each plate.

DETAILED DESCRIPTION

The following description of particular aspect(s) is merely exemplary innature and is in no way intended to limit the scope of the invention,its application, or uses, which may, of course, vary. The disclosure isdescribed with relation to the non-limiting definitions and terminologyincluded herein. These definitions and terminology are not designed tofunction as a limitation on the scope or practice of the disclosure butare presented for illustrative and descriptive purposes only.

The terminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting. As used herein, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms, including “at least one,” unless the content clearly indicatesotherwise. “Or” means “and/or.” As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. It will be further understood that the terms “comprises”and/or “comprising,” or “includes” and/or “including” when used in thisspecification, specify the presence of stated features, regions,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,regions, integers, steps, operations, elements, components, and/orgroups thereof. The term “or a combination thereof” means a combinationincluding at least one of the foregoing elements.

Various embodiments generally provide a composition that decreasesexpression of a CD274 gene or a PD-L1 protein and increases expressionof a PDCD1 gene or a PD-1 protein in a subject. In certain embodiments,the subject is a cell, for example, a tumor cell. More particularly, thecompositions and processes of various embodiments decrease expression ofa CD274 gene or a PD-L1 protein and increase expression of a PDCD1 geneor a PD-1 protein in a subject and are useful for treatment of a tumorwith reduced or even without cardiac side effects. In variousembodiments, the composition includes Type-A polymers including A-Typesingly or doubly linked procyanidin oligomers of the catechins orepicatechins that have utility for altering the expression level of aCD274 gene expression product(s) or a PD-L1 protein, a PDCD1 geneexpression product(s) or a PD-1 protein in a subject, including a cell.

Processes are provided for inhibiting expression of a CD274 gene and/ora PD-L1 protein while enhancing expression of a PDCD1 gene and/or a PD-1protein in a subject, optionally a cell. In some embodiments, processesare provided for inhibiting expression of a CD274 gene and/or a PD-L1protein while enhancing expression of a PDCD1 gene and/or a PD-1 proteinin a cell of a subject. As used herein, a “subject” is defined as anorganism (such as a human, non-human primate, equine, bovine, murine, orother mammal), or a cell. Illustrative examples of cells include tumorcells, such as C6 glioma cells and human macrophage-like THP-1 cells. Asused herein, a subject in need is defined as a subject that hasincreased cellular levels of a CD274 gene and/or a PD-L1 proteinrelative to normal cellular levels and/or decreased cellular levels ofPDCD1 gene and/or a PD-1 protein relative to normal cellular levels, orthat desires to have decreased cellular levels of a CD274 gene and/or aPD-L1 protein relative to normal cellular levels or the subject's owncellular baseline levels and/or increased levels of PDCD1 gene and/or aPD-1 protein relative to normal cellular levels or the subject's owncellular baseline levels.

Moreover, in some embodiments, the composition may be used tosimultaneously inhibit expression of a CD274 gene and/or a PD-L1 proteinwhile enhancing expression of a PDCD1 gene and/or a PD-1 protein. Suchadministration may be used to ameliorate, prevent, or modulate cardiacevents in the subject.

The term “inhibiting” is defined as a decrease in expression, activity,or effect relative to a control related to the presence of an effectorsuch as a Type-A polymer or a component thereof, such as A-Type doublylinked procyanidin oligomers of the catechins and/or epicatechins, aswill be described in greater detail below. Illustrative examples of“inhibiting” are decreases in the cellular expression level or rate ofone or more genes that encode PD-L1 such as CD274, or decreases inlevels of a PD-L1 protein.

The term “enhancing” as used herein is defined as an increase inexpression, activity, or effect relative to a control related to thepresence of an effector such as a Type-A polymer or a component thereof,such as A-Type doubly linked procyanidin oligomers of the catechinsand/or epicatechins. Illustrative examples of “enhanced” are increasesin the cellular expression level or rate of one or more genes thatencode a PD-1 such as PDCD1, or increases in cellular levels of a PD-1protein.

“Active ingredient” refers a component present in the composition thatrenders, directly or indirectly, the intended effect. One particularexample is a polyphenol type-A polymer, with more particular examplesbeing singly linked type-A polymers and/or doubly linked type-Apolymers. Other particular examples include cinnamtannin D-1 and/orcinnamtannin B-1. In some embodiments, the term “active ingredient”excludes singly linked Type-A polymers.

“Polyphenol” as used herein refers to a group of chemical substancesfound in plants, characterized by the presence of more than one phenolgroup per molecule. For purposes of this disclosure, it is to beunderstood that polyphenols include, but are not limited to, Type-Apolymers and oligomers or phenolic materials. Natural sources ofpolyphenols include green tea, white tea, red wine, dark chocolate,olive oil, and other fruits, vegetables, and plants including cinnamon.

In various embodiments, the composition includes at least 0.5% Type-Apolymers by dry weight. Type-A polymers may include A-Type singly and/ordoubly linked procyanidin oligomers of the catechins and/orepicatechins. Such A-Type singly linked procyanidin oligomers of thecatechins and/or epicatechins can include A-Type singly linkedprocyanidin dimers, A-Type singly linked procyanidin trimers, A-Typesingly linked procyanidin trimers, A-Type singly linked procyanidintetramers, and/or a mixture of A-Type singly linked procyanidinoligomers. Such A-Type doubly linked procyanidin oligomers of thecatechins and/or epicatechins can include A-Type doubly linkedprocyanidin dimers, A-Type doubly linked procyanidin trimers, A-Typedoubly linked procyanidin trimers, A-Type doubly linked procyanidintetramers, and/or a mixture of A-Type doubly linked procyanidinoligomers. In various embodiments, the A-Type doubly linked procyanidinoligomers of the catechins and/or epicatechins include cinnamtannin D-1and/or cinnamtannin B-1.

Type-A polymers as used herein are a bioactive type of naturallyavailable polymers. They are identified by their protonated molecularmasses as A-type singly or doubly linked procyanidin oligomers of thecatechins and/or epicatechins. The polymers are composed of monomericunits of catechins and/or epicatechins with a molecular mass of 288 Da.A-type doubly linked procyanidin oligomers may have masses ranging from576 to 1728 Da and may include dimers, trimers, tetramers, and a mixtureof oligomers, respectively. For example, two separate doubly linked TypeA trimers and a doubly linked Type A tetramer have molecular masses of864 and 1152 Da, respectively. The trimer and tetramer oligomers includeterminal (T), middle (M) and base (B) units, with the M unit of the twotrimers consisting of a single catechin/epicatechin and the M unit ofthe tetramer consisting of two catechins/epicatechins. Doubly linkedprocyanidin type-A oligomers of the catechins and/or epicatechinscontain C4→C8 carbon and C2→O7 ether bonds between the T and M units ofthe oligomers, and have the structure:

(Anderson et al., J. Agric. Food Chem., 2004; 52:65-70.) Thus, incertain embodiments, Type-A polymers can include A-type singly or doublylinked procyanidin dimers of catechins and/or epicatechins, A-typesingly or doubly linked procyanidin trimers of catechins and/orepicatechins, A-Type singly or doubly linked procyanidin tetramers ofcatechins and/or epicatechins, and/or a mixture of A-Type singly ordoubly linked procyanidin oligomers of catechins and/or epicatechins. Inother embodiments, Type-A polymers can include A-Type doubly linkedprocyanidin dimers of catechins and/or epicatechins, A-type doublylinked procyanidin trimers of catechins and/or epicatechins, A-Typedoubly linked procyanidin tetramers of catechins and/or epicatechins,and/or a mixture of A-Type doubly linked procyanidin oligomers ofcatechins and/or epicatechins. In some embodiments, the Type-A polymerscan include cinnamtannin D-1 and/or cinnamtannin B-1. In someembodiments, A-Type singly linked procyanidin oligomers of the catechinsand/or epicatechins are excluded from a composition.

In some embodiments, the Type-A polymers can include cinnamtannin D1((2R,3R,4S,8S, 14R,15R)-2,8-bis(3,4-dihydroxyphenyl)-4-[(2R,3S)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-1-benzopyran-8-yl]-3,4-dihydro-2H,8H,14H-8,14-methano-1,7,9-trioxabenzo[6,7]cycloocta[1,2-a]naphthalene-3,5,11,13,15-pentol)and/or cinnamtannin B1. Cinnamtannin D1 has the following structure:

Cinnamtannin B1 has the following structure:

In some embodiments, the amount of active ingredient (e.g., cinnamtanninD1 or cinnamtannin B1) is in the range of 0.5% to 25%, optionally 1% to10% by weight. For example, the amount of active ingredient is greaterthan or equal to 0.5%, greater than or equal to 1%, greater than orequal to 2%, greater than or equal to 3%, greater than or equal to 4%,greater than or equal to 5%, greater than or equal to 10% by weight. Insome embodiments, an active ingredient is or is a part of a broadercomposition. For example, the active ingredient may be present in thebroader composition at 0.5%-100% by weight, 1%-50% by weight, 1%-10% byweight, 20%-50% by weight, 30%-40% by weight, or any value or rangebetween 0.5% and 100% of the dry weight of the broader composition.

In some embodiments, the amount of polyphenol Type-A polymers optionallydoubly linked type-A polymers or the like is in the range of 0.5% to25%, optionally 1% to 10% by weight. For example, the amount ofpolyphenol Type-A polymers is greater than or equal to 0.5%, greaterthan or equal to 1%, greater than or equal to 2%, greater than or equalto 3%, greater than or equal to 4%, greater than or equal to 5%, orgreater than or equal to 10% by weight. In embodiments in which theType-A polymers are doubly-linked polyphenol Type-A polymers, the amountof doubly-linked polyphenol Type-A polymers is in the range of 0.5% to25%, optionally 1% to 10% by weight. For example, the amount ofdoubly-linked polyphenol Type-A polymers may be greater than or equal to0.5%, greater than or equal to 1%, greater than or equal to 2%, greaterthan or equal to 3%, greater than or equal to 4%, greater than or equalto 5%, or greater than or equal to 10% by weight. In some embodiments, aType-A polymer is or is a part of a dietary supplement composition. AType-A polymer is optionally present in a dietary supplement compositionat 0.5%-100% by weight, optionally 1%-50% by weight, optionally 1%-10%by weight, optionally 20%-50% by weight, optionally 30%-40% by weight,or any value or range between 0.5% and 100% of the dry weight of thedietary supplement composition.

Type-A polymers such as A-Type singly and/or doubly linked procyanidinoligomers of the catechins and/or epicatechins may be chemicallysynthesized or obtained from one or more natural sources. One or more ofany process for isolating chemical material from a plant may be used toisolate the Type-A polymers such as A-Type singly and/or doubly linkedprocyanidin oligomers of the catechins and/or epicatechins. Oneexemplary source of type-A polymers is cinnamon. Cinnamon may beobtained from various resources. Illustratively, cinnamon is obtainedfrom bark. Cinnamon bark may be obtained from various parts of theworld, including China, Sri Lanka, Indonesia and others. The type-Apolymers are optionally obtained by carefully tailored extractionprocedures from cinnamon bark. An extract of cinnamon is optionallyderived from any Cinnamonum species. In an exemplary aspect, an extractof cinnamon is derived from the bark of Cinnamonum aromaticum,Cinnamonum verum, or Cinnamomum burmannii. In some aspects, an extractof cinnamon is derived from the bark of the Cinnamomum zeylanicum treeof the genus Lauraceae. This tree is native to eastern and southeasternAsia. Other sources of cinnamon may also be used in the methods andmaterials disclosed herein. Cinnamon bark may be used in the form of rawbark, sliced, or minced bark, or pulverized bark for the preparation ofthe therapeutic materials, and pulverized cinnamon bark is used inparticular instances.

Extracts may be prepared by various methods carefully tailored toproduce the required concentration or amount of type-A polymers. Theextracts are optionally water soluble. As such, the extracts areoptionally water soluble water extracts, water soluble alcohol extracts,or water soluble extracts of other operative extraction processes. Theextraction process is directly linked to the final composition of theresulting extract. As such, a product formed by one process does notnecessarily equate to an extract formed by a different process, oftendiffering by a single extraction parameter. The processes describedherein represent exemplary methods to produce extracts with the desiredlevel of the active agent—Type-A polyphenols, particularly doubly linkedType-A polymers.

Extraction parameters such as water quality, heating temperature, dryingtemperature, heating time, drying time, and filtering processes allcontribute to the quality and efficiency of the processes. Water qualitydirectly affects the concentration of active agents. Poor quality watermay cause type-A polymers to become decomposed and oxidized during theextraction process. This often results in cinnamon extract powder beingreddish in color and the percent concentration of type-A polymers beinglow. Heating time determines the ratio of various polymers beingextracted. Heating time also affects the thickness of extraction mixturewhich then has a direct impact on the downstream filtering process. Thetemperature of the extraction also affects the level of active type-Apolymers. In some aspects, the extraction temperature is between 50° C.and 100° C. Optionally, the extractions temperature is between 50° C.and 95° C. Optionally, the temperature is between 50° C. and 90° C.Optionally the extraction temperature is between 50° C. and 90° C.Drying temperature may vary from 75° C. to 120° C. depending on whatother extraction parameters are also used. The amount of solvent used isgenerally from 2 to 100 times the raw extract material on a weightbasis. Illustratively, when 50 g of cinnamon bark is used, theextraction is performed with 1000 ml of water (1 g/ml is weight ofwater—i.e. 20 times volume).

Extraction time is also important for obtaining the desired amount of,polyphenol Type-A polymers, which are described in detail above.Extractions are optionally performed by heating the raw material in anextraction solvent in excess of 10 minutes, optionally, in excess of 1hour, optionally between 1 and 3 hours with any subdivision alsooperable.

Extraction solvents are optionally aqueous or organic. Distilled wateror alcohols such as ethanol are optionally used alone or in combinationas extraction solvents. The extracts obtained are optionally watersoluble.

Achieving the necessary therapeutic effects of an extracted polyphenolcomposition requires steps beyond simple water or alcohol extraction. Insome aspects, liquid extracts are further processed by columnchromatography to further isolate the actives, optionally by molecularsieve column appropriately sized to isolate the active.

Illustrative examples of cinnamon extracts that contain the requisiteamount of type-A polymers are found in U.S. Pat. No. 6,200,569, which ishereby incorporated by reference in its entirety, and therein describingthe product sold as CINNULIN PF. Optionally, doubly linked procyanidintype-A polymers (3% by weight) in a dried extract of bark from C.burmanni may be prepared as described (Anderson et al., J. Agric. FoodChem. 2004; 52:65-70), or provided by IN Ingredients Inc. (formerlyIntegrity Nutraceuticals, Columbia, Tenn., USA) as CINNULIN PF.

In some embodiments, 50 g clean cinnamon bark is ground into smallparticles or powder. The powder or particles are mixed with 1000 mldistilled water in a suitable flask. The mixture is let stand at roomtemperature for about 0.5 hour. In this and other examples, an amount ofbuffer is optionally added to maintain the pH of the extraction solvent.Additional water may be added is in the range of 1:20 to 1:2000. Toolittle water may render the mixture too thick for extraction. However,too much water increases drying time. Then the water mixture is heatedwhile being stirred through the use of a magnetic heat stirrer. Thetemperature and extraction time are crucial to the concentrationefficiency of the bioactive polymers. The extraction process isoptionally no longer than one hour. Optionally, the ground bark may beheated for 15-20 minutes bringing to a boil, simmering for 20-30 minuteswhile stirring constantly. Optionally, the ground bark is heated to 100°C. 15-20 minutes and then simmered for 20-30 minutes while stirringconstantly. The boiling time is optionally controlled at about 20-25minutes following heating. The mixture is cooled and stored at 4° C.overnight. An exemplary cinnamon extract obtained by a water extractionis sold as CINNULIN PF.

In one exemplary embodiment, 250 kg of Cinnamomum burmannii, is groundinto small particles or powder. The powder or particles are mixed with2000 ml (8×) distilled ethanol-water in a suitable flask and the mixtureis allowed to stand at ambient temperature for 0.5 hours. Optionally,water alone is used as the extraction solvent illustratively by using a10× fold-water volume/weight ground cinnamon bark. The mixture is heatedto 50° C. while being stirred through the use of a magnetic heat stirrerand circulated for 120 min. Evaporation is performed at a steamtemperature of less than 100° C. with a process temperature of less than60° C. with a TS refract meter of 45-50%. The liquid extract is filteredand further purified as above. The material is then dried to a moisturecontent of less than 5%.

In some exemplary embodiments, Type-A polyphenols are extracted fromcinnamon using the following process: 5 g cinnamon and 100 ml 0.1 Nacetic acid are combined and autoclaved for 15 minutes. The resultantmixture is cooled, then centrifuged and the precipitate discarded. Fourvolumes of ethanol/0.1 N acetic acid are added to the supernatant andthe mixture is stored overnight at 4 Co. The mixture is screened througha filter. To determine the amount of bioactive polymers the mixture isintroduced onto an LH-20 column and washed with 600 ml ethanol/0.1 Nacetic acid. The desired fraction is then eluted with a 1:1 mixture ofacetonitrile and 0.2 N acetic acid. The eluate is then concentrated andintroduced onto a HPLC column at 275 nm.

In some embodiments, the initial extraction is performed in the absenceof acid. 50 g clean cinnamon bark is ground into small particles orpowder and mixed with 1000 ml distilled water/10% ethanol in a suitableflask. Then the water mixture is heated while being stirred through theuse of a magnetic heat stirrer. The extraction process is optionally nolonger than one hour. Optionally, the ground bark in extraction solventis heated to a boil for 15-20 minutes, and then simmered for 20-30minutes while stirring constantly. The boiling time is typicallycontrolled at about 20-25 minutes following heating. The mixture iscooled and stored at 4° C. overnight. It is appreciated that alcoholsother than or in addition to ethanol, illustratively methanol, may beused in the extraction procedure as well. When alcohol is used in theextraction solvent is it generally present at 50% or less.

Any one of the extraction solutions (or combinations thereof) describedherein is optionally filtered through a filter paper to remove any soliddebris. If the solution is too thick for the filter paper, the removalof solids from the solution is optionally done with the use ofcentrifugation. The resulting supernatant is filtered through mediumspeed filter paper. The resulting solids are optionally dissolved in 200mL distilled water, or water/ethanol for a second extraction. The liquidsolution containing the solids is mixed and heated for 30 minutes at80-90° C. and then is filtered to produce a second extraction solution.

In some embodiments, first and second extraction solutions are combinedtogether and poured onto nonstick tray and allowed to dry at 80-90° C.Vacuum-spray dry equipment is optionally used for the drying procedure.The resulting dry extract powder is weighed. An extraction ratio iscalculated as w/20×100% with w as the weight (g) of the dry extractpowder. The sample and water ratio, heat time, volume of water in thesecond extraction may vary depending on the amount of the raw materialused for extraction.

High performance liquid chromatography (HPLC) is optionally employed toanalyze the effect on the concentrations of the polymers by changes inheating temperature and extraction time. As a non-limiting example, 100mg dry cinnamon powder is dissolved with 100 ml water in a flask. Thesolution is sonicated for 30-45 minutes and filtered through 0.45 μmPTFE syringe. The samples are prepared and tested at differenttemperatures as follows: samples are extracted at 50-60° C. for onehour, Type-A polymers eluting at 17 and 21 minutes have reasonableconcentrations. After increasing the temperature to 75-82° C. for 1hour, the peaks eluting at 17 and 21 minutes are decreased by 2-3%.There are additional two relatively small peaks that seem to surfaceduring this extraction. They elute at 28.5 minutes, 33.5 minutesrespectively. After the heating temperature is increased to 85-90° C.for an additional 1 hour, the peaks eluting at 17 and 21 minutes aredecreased about 7-9%. The peaks at 28.5 and 33.5 increase significantly.Lastly, the heating temperature is increased to 95-100° C. for 20minutes and then reduced to 85-95° C. for an additional 40 minutes. Thepeaks eluting at 17 and 21 minutes seem to decrease by 15-20%. The peakseluting at 28.5 and 33.5 minutes increase by more than double. Accordingto these results, the polymers at 17 and 21 minutes are converted toisomers at 28.5 and 33.5 minutes respectively.

In another procedure, the stabilization of the Type-A polymers isanalyzed. Various extraction periods at heating temperature of 50-100°C. are tested particularly 95-100° C. After samples are extracted at50-100° C. for one hour, polymer eluting at 17 and 21 minutes presentsdesirable concentrations. The peaks eluting at 17 and 21 minutesdecrease as the heating temperature increases in the first 2-3 hours.After 3 hours, the peaks eluting at 17 and 21 minutes no longer changeas significantly and seem to reach a plateau period. These resultssuggest that after a 3 hour extraction time at temperature of 95-100°C., polymers are stabilized.

Not only is it important to note that the time and temperature play akey factor in sustaining higher concentrations of these Type-A polymerkey actives, additionally the species of choice can have a dramaticimpact on the levels of these Type-A polymers. After thorough review ofthe world's many species of cinnamon, the following has proven toprovide the highest level of active Type-A polymers: CinnamomumBurmannii (Nees) Blume—Microbial Identification Index (MIDI) class;Korintji Cassia.

Cinnamon extract dry powder prepared as discussed above is tested toconfirm the presence of certain amount of polyphenols such asdouble-linked polyphenol Type-A polymers singly-linked Type-A polymers,or other bioactive polymers through the use of HPLC. In embodiments, theType-A polymers may include cinnamtannin D-1 and/or cinnamtannin B-1.This allows for standardization of the extract.

In particular instances, the dry weight of the cinnamon extract powdercan be standardized on the basis of a bioactive component, such asdoubly-linked polyphenol Type-A polymers. The amount of polyphenolType-A polymers or the like is optionally in the range of 0.5% to 25%,optionally 1% to 10% by weight. Optionally, the amount of polyphenolType-A polymers is greater than 0.5%, greater than 1%, greater than 2%,greater than 3%, greater than 4%, greater than 5%, or greater than 10%by weight. In further aspects, the amount of doubly-linked polyphenolType-A polymers or the like is optionally in the range of 0.5% to 25%,optionally 1% to 10% by weight. Optionally, the amount of doubly-linkedpolyphenol Type-A polymers is greater than 0.5%, greater than 1%,greater than 2%, greater than 3%, greater than 4%, greater than 5%, orgreater than 10% by weight.

Depending on the source material, extraction procedures, extractionsolvents, purification and concentration steps, etc., the finalconcentration of Type-A polymers is often insufficient or less than0.5%, less than 1%, less than 2%, less than 3%, less than 4%, less than5%, or less than 10% by weight. As such, the extract may be furtherprocessed to concentrate the type-A polymers to the desired or necessaryconcentration. A liquid extract is optionally passed over a column toprovide a concentrated eluant with the target concentration of type-Apolymers.

Cinnamon bark may be used in the form of raw bark, sliced, or mincedbark, or pulverized bark for the preparation of the therapeuticmaterials, and pulverized cinnamon bark is used in particular instances.

In one experimental series, an extract is prepared according to theforegoing procedures using a water extraction solvent. The concentrationof the sample is approximately (e.g. within error) 5.17 mg/ml. It isalso very important to note that the concentrations of the polymerschange with the temperature and extraction time.

In various embodiments, other natural compounds may be used as an activeingredient. For example, garlic acid, rutin, quercetin, olive leafextract, rosemary extract, black chokeberry extract from Aronia, and/orcurcumin extract may be used as the active ingredient in thecomposition. Garlic acid is a trihydroxybenzoic acid, a type of phenolicacid, found in gallnuts, sumac, witch hazel, tea leaves, oak bark, andother plants. Rutin is a flavonoid, and is one of the polyphenolicscompounds found in vegetables, fruits, herbs, leaves, seeds and severalmedicinal plants. Rutin is a bioflavonoid and antioxidant. Quercetinprotects cell DNA, is anti-inflammatory, a natural histamine blocker,and has the ability to “steal” iron from cancer cells, which can stoptheir growth. Olive leaf extract includes the phytochemical known asoleuropein. Curcumin has powerful antioxidant and anti-inflammatoryproperties.

The rosemary extract may be obtained from various resources. Rosemary,or Rosmarinus officinalis, is a woody bush native to the Mediterraneanregion. Extracts of rosemary may be made from Rosmarinus spp. andpreferably from the leaves and young flowering tops of fresh rosemary(Rosmarinus officinalis L. and its cultivars). Rosemary extraction maybe performed by harvesting the leaves of a rosemary plant and reducingthem in size such as by chopping to improve solvent penetration. Atypical particle size is optionally 0.5-5.0 mm, or any value or rangetherebetween. In some embodiments, the leaf is chopped into a powdertype substance with a particle size of less than 0.5 mm. The choppedplant material combined with a suitable extraction solvent such as wateror a low molecular weight alcohol such as ethanol. The plant material iscombined with the solvent for an extraction time of 18 to 36 hours. Theextraction temperature is optionally the range 10° C. to 45° C. Theresulting extract liquid is separated from the solid material andfiltered, optionally with a sterile filter. Optionally, the resultingextract is poured onto nonstick tray and allowed to dry at 80-90° C.Vacuum-spray dry equipment is optionally used for the drying procedure.The resulting dry extract powder is weighed. An extraction ratio iscalculated as w/20×100% with “w” as the weight (g) of the dry extractpowder. The sample and water ratio, heat time, volume of water in thesecond extraction may vary depending on the amount of the raw materialused for extraction.

Depending on the intended mode of administration, the compositionadministered can be in pharmaceutical compositions in the form of solid,semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, oil based forms, orsuspensions, and may be provided in unit dosages suitable for a singleadministration. Time release preparations are specifically contemplatedas effective dosage formulations. The compositions will include aneffective amount of the selected substrate in combination with apharmaceutically acceptable carrier and, in addition, may include othermedicinal agents, pharmaceutical agents, carriers, or diluents.

In a solid composition embodiment, conventional nontoxic solid carriersmay include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose,sucrose and magnesium carbonate. Liquid pharmaceutically administrablecompositions may, for example, be prepared by dissolving or dispersingan active agent with optimal pharmaceutical adjuvants in an excipient,such as water, saline, aqueous dextrose, glycerol, or ethanol, to form asolution or suspension. For example, the pharmaceutical composition maycontain minor amounts of nontoxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents, for example, sodium acetateor triethanolamine oleate. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington's The Science and Practice of Pharmacy (20^(th)Edition).

In oral administration embodiments, fine powders or granules may containdiluting, dispersing, or surface active agents. The fine powders orgranules may be presented in water or in syrup, in capsules or sachetsin the dry state, or in a non-aqueous solution or suspension. Suspendingagents may also be included in tablets, which may include binders andlubricants in a suspension. Flavoring, preserving, suspending,thickening, or emulsifying agents may be also included to modify thetaste and texture of the composition. The tablets and granules providedfor oral administration may further be coated for ease of digestion.

In some embodiments, the composition containing the active Type-Apolymers may be combined with one or more supplementary active agents. Asupplementary active agent optionally functions synergistically with aType-A polymer. Supplementary active agents illustratively includevitamins (such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin Eor vitamin K), antioxidants (such as acai, wolfberry, alpha lipoic acid,astazanthin, or fucoxanthin), or any combination of the above.

The composition including Type-A polymers is optionally in the form of afood additive. Examples include foods in a liquid, semi-liquid, solid,paste, or jelly form.

Compositions may be metabolized in the subject to yield atherapeutically effective amount of compound species, namely polyphenolssuch as a Type-A polyphenols as discussed in detail above, cinnamonoligomer, cinnamon catechin or epicatechin, cinnamon chalcone, andcinnamon MHCP. In particular therapies, each dose of the cinnamonextract supplement is selected so as to deliver into the individualType-A polymers in the amount of 0.1 milligrams (mg) to 150 mg of Type-Apolymer per serving or any value or range therebetween, optionally 1-30mg, and optionally 3-10 mg.

In an exemplary regimen, the composition is taken orally between one andthree times daily. However, it is contemplated that other routes ofadministration may be utilized. Also, it should be noted that thecompositions may be utilized in the form of derivatives. For example,the composition may be bonded, chemically or physically, to otherchemical species and moieties such as synthetic polymers, liposomes,small organic molecules, chitin, chitosan, other biopolymers and thelike. In view of the teaching presented herein, still furthercombinations will be readily apparent to those of skill in the art.

In various embodiments, a subject is administered a composition in adosage so that each dose of the compositions is selected to deliver intothe Type-A polymers in the amount of 0.1 milligrams (mg) to 150 mg ofType-A Polymer per serving or any value or range therebetween,optionally 1-30 mg, optionally 3-10 mg. It is further contemplated thatvariable dosing regiments are operative in the processes. While in someinstances, a single dose treatment may be effective in producingtherapeutic or other desired effects, in other instances a treatmentperiod in the range of, for example, six weeks to three or six months ormore may be utilized.

The composition may be administered orally, parentally, or intravenouslyby intramuscular, intraperitoneally, by transdermal injection, or bycontact with a cell or tissue such as by immersion or other form ofcontact. Injectable or oral dosage forms may be prepared in conventionalforms, either liquid solutions or suspensions, solid forms suitable forsolution or prior to administration, or as suspension in liquid prior toadministration or as emulsions.

Additionally, the dosage forms may be an oil-based form. The dose of thecomposition may vary depending on the age, weight, general condition ofthe subject. For example, dosage in the range of 1-1,000 mg orequivalent of at least 0.5% Type-A polymers by dry weight per day may bean effective range. The active agent may be present at 0.01%-100% of thedry weight of the composition. For example, an active agent may comprise0.5%-50% of the dry weight of the composition.

Administration of a composition to a subject (e.g., a cell) will inhibitexpression of a PD-L1 gene or protein in a subject (e.g., in a cell)relative to control or baseline. Illustratively, a PD-L1 gene expression(such as CD274) is decreased as measured by the level of PD-L1 mRNA(such as CD274 mRNA) relative to a control such as the absence ofcomposition. Illustratively, PD-L1 gene expression is inhibited (e.g.,decreased) by a value of 1% to 300% or more, or any value or rangetherebetween. Optionally, PD-L1 gene expression is decreased by 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or more.

The level of target of PD-1 gene expression (such as PDCD1) may beenhanced (e.g., increased) in the subject (e.g., the cell) relative tocontrol or baseline. Illustratively, expression of the gene encodingPD-1 (PDCD1) is enhanced by a value of 1% to 300% or more, or any valueor range therebetween. Optionally, expression of the gene encoding PD-1is enhanced by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250%,300%, or more relative to control.

Methods for detecting mRNA expression to determine the presence orextent of gene expression are known in the art. Illustratively, mRNA isdetected and optionally quantified by real-time polymerase chainreaction (qRT-PCR as used herein). qRT-PCR is optionally coupled toprior synthesis of cDNA from total cellular RNA such as usingSuperscript II RT which is a reverse transcriptase enzyme produced byInvitrogen, Corp., Carlsbad, Calif. Illustrative protocols for measuringgene expression can be found in Crujeiras A B, et al., Eur J ClinInvest, 2008; 38(9):672-8, as well as in other sources known in the art.

Expression of a PD-L1 protein in a subject (e.g., a cell) is optionallyinhibited (i.e. decreased) by administration of a composition to asubject. Illustratively expression of the PD-L1 protein is inhibited(e.g., in a cell) relative to a control or baseline. Illustratively,PD-L1 protein expression is inhibited by a value of 1% to 300% or more,or any value or range therebetween. Optionally, PD-L1 protein expressionis inhibited by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%,250%, 300%, or more.

Expression of a PD-1 protein in a subject (e.g., a cell) may be enhanced(i.e., increased) by administration of the composition to the subject.In some embodiments, expression of the PD-1 protein is enhanced relativeto a control or baseline. For example, PD-1 protein expression may beenhanced by a value of 1% to 300% or more, or any value or rangetherebetween. Optionally, PD-1 protein expression is enhanced by 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, or more.

Detecting and optionally quantifying PD-L1 protein or PD-1 proteinexpression is achieved by many methods known in the art. Illustratively,PD-L1 protein or PD-1 protein expression is detected and optionallyquantified by enzyme linked immunosorbent assay (ELISA), massspectrometry, western blot, gel electrophoresis optionally coupled withstaining such as by Coomassie brilliant blue or silver stain, or bytarget specific stains, flow cytometry, immunoprecipitation, or by othermethod known in the art. In some embodiments, an ELISA is used to detectand optionally quantify PD-L1 protein or PD-1 expression. For example,ELISA kits for PD-L1 or PD-1 are available from sources known in theart. Antibodies directed to PD-L1 or PD-1 proteins suitable for use inELISA are available from sources known in the art, including Santa CruzBiotechnology, Santa Cruz, Calif.

It is appreciated that any dietary supplement or any extract of cinnamondescribed herein or their equivalents are optionally used in a processto treat dysfunctions of the PD-1/PD-L1 system in a subject (e.g., in acell of a subject), including dysregulated PD-L1 levels and particularlyhigh PD-L1 levels, dysregulated PD-1 levels and particularly low PD-1levels, or combinations thereof, as well as symptoms in a subject causedby such dysfunctions of the PD-1/PD-L1 system in a subject (e.g., in acell of a subject). For example, the dietary supplement or any extractof cinnamon described herein or their equivalents are optionally used ina process to treat a tumor with reduced or even without cardiac sideeffects.

A process of treating dysfunctions of the PD-1/PD-L1 pathway in asubject, including dysregulated PD-L1 levels and particularly high PD-L1levels, dysregulated PD-1 levels and particularly low PD-1 levels, andcombinations thereof, is also provided. Such processes illustrativelyinclude administering to a subject a therapeutically effective amount ofa composition including one or more Type-A polymers. A therapeuticallyeffective amount is defined as that capable of decreasing the expressionof a PD-L1 protein or a gene encoding a PD-L1 protein and/or increasingthe expression of a PD-1 protein or a gene encoding a PD-1 protein in asubject (e.g., in a cell) relative to a control.

Processes of treating dysfunctions of the PD-1/PD-L1 pathway in a cell,including dysregulated PD-L1 levels and particularly high PD-L1 levels,dysregulated PD-1 levels and particularly low PD-1 levels, andcombinations thereof, illustratively include administering to a subjecta dietary supplement composition including Type-A polymers in a dosageso that each dose of the composition will deliver into the individualType-A polymers in the amount of 0.1 milligrams (mg) to 150 mg of Type-APolymer per serving or any value or range therebetween, such as 1-30 mg,or 3-10 mg.

A process of treating symptoms implicated by dysfunctions of thePD-1/PD-L1 pathway (such as dysregulated PD-L1 levels and particularlyhigh PD-L1 levels and/or dysregulated PD-1 levels and particularly lowPD-1 levels, and combinations thereof) in a cell, including symptoms ofcancer of a subject is also provided. Such processes illustrativelyinclude administering to a subject a therapeutically effective amount ofType-A polymers.

A process of treating symptoms implicated by dysfunctions of thePD-1/PD-L1 pathway (such as dysregulated PD-L1 levels and particularlyhigh PD-L1 levels and dysregulated PD-1 levels and particularly low PD-1levels) in a cell, including symptoms of cardiovascular events in asubject is also provided. Such processes illustratively includeadministering to a subject a therapeutically effective amount of Type-Apolymers to ameliorate, prevent, or modulate cardiac events in thesubject. For example, administration to a subject optionally reduces oreliminates events of cardiomyopathy, optionally heart failure optionallydue to autoimmune dilated cardiomyopathy, relative to controls or otherPD-L1 specific therapeutics.

Processes of treating symptoms implicated by dysfunctions the PD-1/PD-L1pathway (such as dysregulated PD-L1 levels and particularly high PD-L1levels, dysregulated PD-1 levels and particularly low PD-1 levels, orcombinations thereof) in a subject, including symptoms of cancer, viralinfections, and autoimmune diseases such as autoimmune dilatedcardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis,systemic lupus erythematosus, graft-versus-host disease, type I diabetesmellitus, and rheumatoid arthritis, illustratively include administeringto a subject a composition including Type-A polymers in a dosage so thateach dose of the composition will deliver into the individual Type-Apolymers in the amount of 0.1 milligrams (mg) to 150 mg of Type-APolymer per serving or any value or range therebetween, optionally 1-30mg, optionally 3-10 mg.

It is further contemplated that variable dosing regiments are operativein the methods. While in some instances, a single dose treatment may beeffective in producing therapeutic effects, in other instances atreatment period in the range of, for example, six weeks to three or sixmonths or more may be utilized. The composition may be administeredorally, parentally, or intravenously by intramuscular, intraperitoneal,by transdermal injection, or otherwise by contact with a subject.Injectables or oral forms may be prepared in conventional forms, eitherliquid solutions or suspensions, solid forms suitable for solution orprior to administration, or as suspension in liquid prior toadministration or as emulsions.

The dose of the composition may vary depending on the age, weight,general condition of the subject. For example, dosage in the range of1-1,000 mg of at least 0.5% Type-A polymers by dry weight per day may bean effective range. The Type-A polymers may also comprise 0.01%-100% ofthe dry weight of the composition. For example, a dietary supplementcomposition may comprise 0.5%-50% of the dry weight of the composition.

Various embodiments are illustrated by the following non-limitingexamples. The examples are for illustrative purposes and are not alimitation on any practice of the present invention. It will beunderstood that variations and modifications can be made withoutdeparting from the spirit and scope of the invention.

EXAMPLES

In the present study, human macrophage-like THP-1 cells were used todetermine the effects of active agents.

Immunofluorescence studies were utilized to explore the effects of anactive composition including Type-A polymers on PD-L1 and PD-1 proteins.Doubly linked procyanidin type-A polymers (3% by weight) in a driedextract of bark from C. burmanni was prepared as described (Anderson etal., J. Agric. Food Chem. 2004; 52:65-70), or provided by IN IngredientsInc. (formerly Integrity Nutraceuticals, Columbia, Tenn., USA) asCINNULIN PF. The dried extract composition was solvated in DMSO andstored at −20° C. until use. The antibodies (anti-PD-L1, anti-PD-1) wereall obtained from Santa Cruz Biotech. All other reagents used were ofthe highest grade available in commercial products.

Human monocytic THP-1 cells were purchased from ATCC, Manassas, Va. andwere maintained in RPMI culture medium supplemented with 10% heatinactivated fetal bovine serum, 10 mM Hepes, 0.1 mM MEM non-essentialamino acids, 1 mM sodium pyruvate, and 100 nM penicillin/streptomycin inan atmosphere containing 5% CO₂ and 95% air at 37° C. THP-1 monocyteswere differentiated in macrophages by 48 hour incubation with phorbol12-myristate 13-acetate (PMA, Sigma, P8139) in RPMI media at aconcentration of 10 ng/mL. Cells were incubated with or without thetreatment compounds at dosages indicated below at 37° C. for 24 hours.In certain treatment with LPS studies, the cells were incubated in freshF-12 K medium supplemented with 10 μg/mL LPS and the type-A polymers ata concentration (from 3% extract) of 0 μg/ml, 5 μg/ml, 10 μg/ml, 25μg/ml, or 50 μg/ml for 24 h at 37° C. In other treatment with LPSstudies, the cells were incubated in fresh F-12 K medium supplementedwith 10 μg/mL LPS and cinnamtannin D-1 at a concentration 2.5 μg/ml, 5μg/ml, 10 μg/ml or 25 μg/ml for 24 h at 37° C.

Cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehydefor 10 min at room temperature, followed by permeabilization with 0.3%Triton x-100 for 10 min. After being washed with PBS three times, cellswere incubated for 1 h in PBS containing 10% normal goat serum blockingsolution. The cells were subjected to immunofluorescence staining withthe specific antibodies (human macrophage-like THP-1 cells analyzed forPD-1 (Santa Cruz Biotech)) overnight at 4° C. The cells were then washedwith cold PBS three times for 3 min each, and incubated withAlexa-labeled secondary antibodies (Invitrogen) at room temperature for1 h. The cells were examined by fluorescence microscopy (a NikonTE2000-S microscope, Nikon, Tokyo, Japan). For cell counts, five to tenrandom fields with approximately similar density of cells in each fieldwere selected for analysis in each plate. Fluorescence intensities (withpixel values exceeding five times the standard deviation of thebackground) from these images were semi-quantitatively analyzed bydensitometry (ImageJ software, NIH Image).

The Type-A polymer composition (from 3% extract) produced significantlyenhanced expression of PDCD-1 relative to control in humanmacrophage-like THP-1 cells at all concentrations tested. Thus, the datademonstrates that doubly linked procyanidin type-A polymerssignificantly enhance expression of PDCD-1 in cells. Further, the Type-Apolymer composition significantly improved LPS-induced PDCD-1 downexpression at all concentrations tested. Thus, the data demonstratesthat doubly linked procyanidin type-A polymers significantly increaseexpression of PDCD-1 in cells.

As depicted in FIG. 1, the isolated doubly linked type-A polymers asdescribed in Anderson et al., J. Agric. Food Chem., 2004; 52:65-70,which includes cinnamtannin B-1 and cinnamtannin D-1, significantlyenhanced PDCD-1 expression in human macrophage-like THP-1 cells at allconcentrations tested (5 μg/ml, 10 μg/ml, 25 μg/ml, or 50 μg/ml). Ascompared to controls, Cinnulin PF resulted in a percent increase ofPDCD-1 expression of 13% increase at 5 μg/ml, 29% increase at 10 μg/ml,67% increase at 25 μg/ml, and 58% increase at 50 μg/ml of Cinnulin PF.

As depicted in FIG. 2, the cinnamtannin B-1 significantly enhancedPDCD-1 expression in human macrophage-like THP-1 cells at allconcentrations tested (2.5 μg/ml, 5 μg/ml, 10 μg/ml, or 20 μg/ml). Ascompared to controls, cinnamtannin B-1 resulted in a percent increase ofPDCD-1 expression of 21% increase at 2.5 μg/ml, 42% increase at 5 μg/ml,83% increase at 10 μg/ml, and 62% increase at 20 μg/ml of cinnamtanninB-1.

As depicted in FIG. 3, the cinnamtannin D-1 significantly enhancedPDCD-1 expression in human macrophage-like THP-1 cells at allconcentrations tested (2.5 μg/ml, 5 μg/ml, 10 μg/ml, or 20 μg/ml). Ascompared to controls, cinnamtannin D-1 resulted in a percent increase ofPDCD-1 expression of 18% increase at 2.5 μg/ml, 45% increase at 5 μg/ml,87% increase at 10 μg/ml, and 71% increase at 20 μg/ml of cinnamtanninB-1.

As depicted in FIG. 4, isolated doubly linked type-A polymers asdescribed in Anderson et al., J. Agric. Food Chem., 2004; 52:65-70significantly improved LPS-induced PD-1 down-regulation at allconcentrations tested. As compared to controls, the doubly linked-type Apolymers resulted in a percent inhibition of LPS-induced PDCD-1 downexpression of 27% inhibition at 5 μg/ml, 70% inhibition at 10 μg/ml, 79%inhibition at 25 μg/ml, and 31% inhibition at 50 μg/ml of active.

As depicted in FIG. 5, cinnamtannin D-1 significantly improvedLPS-induced PD-1 down-regulation at all concentrations tested. Ascompared to controls, cinnamtannin D-1 resulted in a percent inhibitionof LPS-induced PDCD-1 down expression of 42% inhibition at 2.5 μg/ml,98% inhibition at 5 μg/ml, 81% inhibition at 10 μg/ml, and 48%inhibition at 25 μg/ml of cinnamtannin D-1.

In addition to cinnamtannins, various other natural compounds weretested to determine if they enhanced PD-1 expression. Compounds includedolive leaf extract, quercetin, garlic acid, rutin, CellBerry® (blackchokeberry extract from Aronia, commercially available from INIngredients Inc. Columbia, Tenn., USA), rosemary extract, and curcuminextract.

As depicted in FIG. 6, the garlic acid significantly enhanced PDCD-1expression in human macrophage-like THP-1 cells at all concentrationstested (10 μg/ml, 20 μg/ml, or 50 μg/ml). As compared to controls,garlic acid resulted in a percent increase of PDCD-1 expression of 47%increase at 10 μg/ml, 71% increase at 20 μg/ml, and 52% increase at 50μg/ml of garlic acid.

As depicted in FIG. 7, the rutin significantly enhanced PDCD-1expression in human macrophage-like THP-1 cells at all concentrationstested (10 μg/ml, 20 μg/ml, or 50 μg/ml). As compared to controls, rutinresulted in a percent increase of PDCD-1 expression of 17% increase at10 μg/ml, 48% increase at 20 μg/ml, and 43% increase at 50 μg/ml ofrutin.

As depicted in FIG. 8, the quercetin significantly enhanced PDCD-1expression in human macrophage-like THP-1 cells at all concentrationstested (10 μg/ml, 20 μg/ml, or 50 μg/ml). As compared to controls,quercetin resulted in a percent increase of PDCD-1 expression of 33%increase at 10 μg/ml, 78% increase at 20 μg/ml, and 57% increase at 50μg/ml of quercetin.

As depicted in FIG. 9, the olive leaf extract significantly enhancedPDCD-1 expression in human macrophage-like THP-1 cells at allconcentrations tested (10 μg/ml, 50 μg/ml, or 100 μg/ml). As compared tocontrols, olive leaf extract resulted in a percent increase of PDCD-1expression of 28% increase at 10 μg/ml, 67% increase at 50 μg/ml, and79% increase at 100 μg/ml of olive leaf extract.

As depicted in FIG. 10, the rosemary extract significantly enhancedPDCD-1 expression in human macrophage-like THP-1 cells at allconcentrations tested (10 μg/ml, 50 μg/ml, or 100 μg/ml). As compared tocontrols, rosemary extract resulted in a percent increase of PDCD-1expression of 35% increase at 10 μg/ml, 53% increase at 50 μg/ml, and89% increase at 100 μg/ml of rosemary extract.

As depicted in FIG. 11, the CellBerry® significantly enhanced PDCD-1expression in human macrophage-like THP-1 cells at all concentrationstested (10 μg/ml, 20 μg/ml, or 100 μg/ml). As compared to controls,CellBerry® resulted in a percent increase of PDCD-1 expression of 23%increase at 10 μg/ml, 35% increase at 20 μg/ml, and 48% increase at 100μg/ml of CellBerry®.

As depicted in FIG. 12, the curcumin extract significantly enhancedPDCD-1 expression in human macrophage-like THP-1 cells at allconcentrations tested (5 μg/ml, 10 μg/ml, or 50 μg/ml). As compared tocontrols, curcumin extract resulted in a percent increase of PDCD-1expression of 21% increase at 5 μg/ml, 45% increase at 10 μg/ml, and 93%increase at 50 μg/ml of curcumin extract.

In the present study of PD-L1 expression, rat C6 glioma cells and humanmacrophage-like THP-1 cells were used to determine the effects of activeagents.

Immunofluorescence studies were utilized to explore the effects of anactive composition including Type-A polymers on PD-L1 proteins. Purifiedcinnamtannin B1 and cinnamtannin D1 are obtained from Planta Analytica(New Milford, Conn.). A water extract of cinnamon with at least 3% byweight type-A polymers was obtained from IN Ingredients sold as CINNULINPF. Each dried material was solvated in DMSO and stored at −20° C. untiluse. The antibodies (anti-PD-L1) were all obtained from Santa CruzBiotech. All other reagents used were of the highest grade available incommercial products.

C6 glioma cells (CCL-107) were purchased from American Type CultureCollection (ATCC; Manassas, Va.). Cell cultures were grown in F-12 Kmedium (Gibco/Invitrogen) supplemented with 10% horse serum and 2% fetalbovine serum and maintained in a humidified atmosphere containing 5% CO₂and 95% air at 37° C. Cultures were grown to 85% confluency in 75 mmflasks, and cells were subcultured by trypsinization of subconfluentcultures using 0.05% trypsin with EDTA. C6 glioma cells were seeded at adensity of 0.5×10⁶ cells per 35 mm dish, and cultured for two days.Cells were grown to confluence during the experimental period. Threestudies were performed: 1) no other treatment of the cells; 2) treatmentwith LPS (10 μg/mL); and 3) treatment with TNF-α (10 ng/mL). In somestudies, cells were incubated in fresh F-12K medium supplemented witheither cinnamtannin D1 (at a concentration of 0 μg/ml, 5 μg/ml, 10μg/ml, 20 μg/ml, 25 μg/ml, or 50 μg/ml) or cinnamtannin B1 (at aconcentration of 0 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 25 μg/ml, or 50μg/ml) for 24 h at 37° C. In certain treatment with LPS studies, thecells were incubated in fresh F-12 K medium supplemented with 10 μg/mLLPS or incubated in fresh F-12 K medium supplemented with 10 μg/mL LPSand cinnamtannin D1 at a concentration 10 μg/ml or 50 μg/ml (5 μg/ml and25 μg/ml were not tested) for 24 h at 37° C. In certain treatment withTNF-α studies, the cells were incubated in fresh FK-12 mediumsupplemented with 10 ng/mL TNF-α and cinnamtannin D1 at a concentration10 μg/ml, 20 μg/ml or 50 μg/ml (5 μg/ml was not tested) for 24 h at 37°C.

Human monocytic THP-1 cells were purchased from ATCC, Manassas, Va. andwere maintained in RPMI culture medium supplemented with 10% heatinactivated fetal bovine serum and 50 pM β-mercaptoethanol in ahumidified atmosphere containing 5% CO₂ and 95% air at 37° C. THP-1monocytes are differentiated in macrophages by 24 hour incubation with150 nM phorbol 12-myristate 13-acetate (PMA, Sigma, P8139). Two studieswere performed: 1) no other treatment of the cells; and 2) treatmentwith LPS (10 μg/mL). For the no other treatment studies, the cells wereincubated in fresh RPMI medium supplemented with the notedconcentrations of extract of 0 μg/ml, 5 μg/ml, 10 μg/ml, or 25 μg/ml for72 h at 37° C. For the treatment with LPS studies, the cells wereincubated in fresh RPMI medium supplemented with 10 μg/mL of LPS and aconcentration of active of 0 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, or 50μg/ml for 24 h at 37° C.

Cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehydefor 10 min at room temperature, followed by permeabilization with 0.3%Triton x-100 for 10 min. After being washed with PBS three times, cellswere incubated for 1 h in PBS containing 10% normal goat serum blockingsolution. The cells were subjected to immunofluorescence staining withthe specific antibodies (Rat C6 glioma cells studied for PD-L1 (SantaCruz Biotech); human macrophage-like THP-1 cells analyzed for PD-1(Santa Cruz Biotech)) overnight at 4° C. The cells were then washed withcold PBS three times for 3 min each, and incubated with Alexa-labeledsecondary antibodies (Invitrogen) at room temperature for 1 h. The cellswere examined by fluorescence microscopy (a Nikon TE2000-S microscope,Nikon, Tokyo, Japan). For cell counts, five to ten random fields withapproximately similar density of cells in each field were selected foranalysis in each plate. Fluorescence intensities (with pixel valuesexceeding five times the standard deviation of the background) fromthese images were semi-quantitatively analyzed by densitometry (ImageJsoftware, NIH Image).

As depicted in FIG. 13, the cinnamtannin D1 significantly inhibitedPD-L1 expression in rat C6 glioma cells at all concentrations tested. Ascompared to controls, cinnamtannin D1 resulted in a percent inhibitionof PD-L1 expression of 39.9 percent inhibition at 5 μg/ml ofcinnamtannin D1, 66.8 percent inhibition at 10 μg/ml of cinnamtannin D1,58.1 percent inhibition at 25 μg/ml of cinnamtannin D1, and 43.6 percentinhibition at 50 μg/ml of cinnamtannin D1.

As depicted in FIG. 14, the cinnamtannin B1 significantly inhibitedPD-L1 expression in rat C6 glioma cells at all concentrations tested. Ascompared to controls, cinnamtannin B1 resulted in a percent inhibitionof PD-L1 expression of 35.7 percent inhibition at 5 μg/ml ofcinnamtannin B1, 60.8 percent inhibition at 10 μg/ml cinnamtannin B1,54.9 percent inhibition at 25 μg/ml of cinnamtannin B1, and 41.9 percentinhibition at 50 μg/ml of cinnamtannin B1.

As depicted in FIG. 15, the cinnamtannin D1 significantly inhibitedTNF-α-induced overexpression of PD-L1 at all concentrations tested. Ascompared to controls, cinnamtannin D1 resulted in a percent inhibitionof TNF-α-induced overexpression of PD-L1 of 32.0 percent inhibition at10 μg/ml of cinnamtannin D1, 42.2 percent inhibition at 20 μg/ml ofcinnamtannin D1, and 43.9 percent inhibition at 50 μg/ml of cinnamtanninD1.

As depicted in FIG. 16, the cinnamtannin D1 significantly inhibitedLPS-induced overexpression of PD-L1 at all concentrations tested. Ascompared to controls, cinnamtannin D1 resulted in a percent inhibitionof LPS-induced overexpression of PD-L1 of 48.9 percent inhibition at 10μg/ml of cinnamtannin D1, and 53.0 percent inhibition at 50 μg/ml ofcinnamtannin D1.

As illustrated in FIG. 17, the Type-A polymer composition (from 3%extract) produced significantly inhibited expression of PD-L1 relativeto control in rat C6 glioma cells at all concentrations tested (5 μg/ml,10 μg/ml, 50 μg/ml, or 100 μg/ml) showing inhibition of expression of10.4%, 21.7%, 58.4%, and 41.7%, respectively. In additional data notshown, the Type-A polymer composition produced significantly inhibitedLPS-induced overexpression of PD-L1 using both 10 μg/ml and 10 μg/mlconcentrations (5 μg/ml and 20 μg/ml was not tested). Additionally, theType-A polymer composition produced significantly inhibitedTNF-α-induced overexpression of PD-L1 using 10 μg/ml, 20 μg/ml, and 50μg/ml concentrations (5 μg/ml was not tested). Thus, the datademonstrates that doubly linked procyanidin type-A polymerssignificantly decrease expression of PD-L1 in cells.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed herein above.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified.

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the inventionpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual application orpublication was specifically and individually incorporated herein byreference for the entirety of their teaching.

The foregoing description is illustrative of particular aspects of theinvention, but is not meant to be a limitation upon the practicethereof.

1. A process of treating an autoimmune disease or condition in a humanin need thereof comprising administering to said human in need thereof acomposition comprising at least 5.0 weight % isolated cinnamtannin D-1,at least 5.0 weight % isolated cinnamtannin B-1, or a combinationthereof to effectively treat the autoimmune disease or condition in saidhuman in need thereof.
 2. The process of claim 1, wherein saidcinnamtannin D-1 and/or cinnamtannin B-1 is present at about 1-30milligrams.
 3. The process of claim 1, wherein said cinnamtannin D-1and/or cinnamtannin B-1 is present at about 3-10 milligrams.
 4. Theprocess of claim 1, wherein said active ingredient consists essentiallyof cinnamtannin D-1 and cinnamtannin B-1.
 5. The process of claim 1,wherein the composition is administered orally, intravenously, byintramuscular injection, by intraperitoneal injection, or transdermally.6. The process of claim 1, wherein said composition further comprisesone or more vitamins, antioxidants, or combinations thereof.
 7. Theprocess of claim 1, wherein said composition is administered daily for aperiod of six weeks or more.
 8. The process of claim 1, wherein saidcomposition is administered one to three times daily.
 9. The process ofclaim 1, wherein the composition is administered orally, intravenously,by intramuscular injection, by intraperitoneal injection, ortransdermally.
 10. The process of claim 1, wherein said compositionfurther comprises one or more vitamins, antioxidants, or combinationsthereof.
 11. The process of claim 1 wherein said autoimmune disease orcondition is selected from the group consisting of autoimmune dilatedcardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis,systemic lupus erythematosus, graft-versus-host disease, type I diabetesmellitus, and rheumatoid arthritis.